The most abundant cell-wall and structural polysaccharide in the plant world is cellulose, a linear polymer of D-glucose with .beta.(1-4) linkages. Cellulose is the principal component of wood and thus of paper; cotton is approximately 72% (by weight) pure cellulose.
On complete hydrolysis with strong acids, cellulose yields only D-glucose, but partial hydrolysis yields mainly the reducing disaccharide cellobiose, in which the linkage between the D-glucose (or anhydroglucose) units is .beta.(1-4), and cellulose oligosaccharides comprised of long, linear chains of anhydroglucose units.
The only chemical difference between starch and cellulose, both homopolysaccharides of D-glucose, is that starch has .alpha.(1-4) linkages and cellulose .beta.(1-4). Enzymes capable of hydrolyzing the .beta.(1-4) linkages of cellulose are not secreted in the digestive tract of most mammals, which therefore cannot use cellulose as food. The ruminants, however, are an exception: they can digest cellulose since bacteria in the rumen form the enzyme cellulase which hydrolyzes cellulose to D-glucose.
Cellulase is an enzyme complex comprising several components, each of which catalyzes the cleavage of a different substrate. A cellulase extract from Trichoderma viride, for example, contains Avicelase, carboxymethyl cellulase, cellobiase, xylanase and salicinase.
Several assay procedures have previously been used to measure the initial degradative steps in cellulose hydrolysis (also called the solubilizing activity) including: the viscosimetric method which uses carboxymethyl cellulose (CM-cellulose) as a substrate, the measurement of glucose or reducing sugars produced from native cellulose, the turbidity measurement of free fiber formation and the dinitrosalicylic acid assay to determine the concentration of reducing sugars released by CM-cellulose.
Each of these procedures, however, has one or more of the following disadvantages: substrate insensitivity, pseudosubstrate recognition, poor correlation between the actual hydrolytic products formed and the products analyzed, discontinuous measurement and interference by common end products (e.g. glucose) or sulfhydryl inhibitors.
The present invention is an improved method for the continuous spectrophotometric assay of cellulase activity in bacteria. The enzymatic degradation of a dyed crystalline cellulose is used to determine cellulase concentrations. Representative of the cellulolytic bacteria suitable for assay according to this invention are Clostridium thermocellum (C. thermocellum) and Trichoderma viride (T. viride). The continuous assay is useful in the study of cellulase kinetics and in the comparative investigation of cellulase activities in microorganisms.
Several dyed cellulose preparations can be used to measure cellulase activity. The invention, however, will hereinafter be described with respect to the use of Remazol Brilliant Blue dyed Avicel-cellulose as illustrative of the dyed cellulose substrates. Dyed-Avicel directly corresponds to the physical characteristics of native cellulose and can be easily prepared.